Corneal blindness affects millions of individuals world-wide and usually can only be treated by transplantation of donated tissue. In many regions of the world donor tissue is not available; in the US, the supply of corneas for transplant is threatened by increasing numbers of eyes with refractive surgeries. The long-term goal of this project is to address corneal blindness by developing therapies based on stem cells. This project has already identified and characterized adult human corneal stromal stem cells (CSSC), which can be expanded in culture while retaining the ability to adopt a keratocyte phenotype and to elaborate abundant stroma-like connective tissue. When CSSC were cultured on a scaffolding of aligned paralel nanofibers of poly(ester urethane)urea (PEUU), a biodegradable polymer, they secreted connective tissue with aligned collagen similar to that of normal corneal stroma. When CSSC were injected into stromas of lumican knockout mice, collagen in the stroma became more organized and corneal transparency was increased. Importantly, human CSSC exhibited an immune privilege in mice, remaining viable for months without immune rejection. The new proposal will test specific hypotheses emanating directly from these exciting findings. Aim 1 documents the process of tissue regeneration by CSSC by testing the predictions that (a) CSSC first remove existing tissue by temporally regulated expression of a specific group of matrix metalloproteinases; (b) after this removal process, CSSC rebuild stromal tissue by deposition of new connective tissue; and (c) this regeneration process can remodel scar tissue similar to that causing most human corneal blindness. Aim 2 combines CSSC with PEUU scaffolding to produce a novel bioengineered tissue and tests the predictions that (a) CSSC monolayers on PEUU nanofiber scaffolding can integrate and function as transparent stromal tissue in mouse corneal transplants; (b) CSSC can be incorporated into multilayered PEUU scaffolding with the thickness and strength of a corneal stroma; and (c) the multilayered constructs can functionally replace stroma after transplantation into rabbit corneas using deep anterior lamellar keratoplasty. The scientific impact of this study will be a demonstration of effectiveness of two novel stem-cell based therapeutic approaches for corneal scarring, indicating a readiness for translation of each to clinical trials.